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Abstract

Background

Ketogenic diets are an effective healthy way of losing weight since they promote a
non-atherogenic lipid profile, lower blood pressure and decrease resistance to insulin
with an improvement in blood levels of glucose and insulin. On the other hand, Mediterranean
diet is well known to be one of the healthiest diets, being the basic ingredients
of such diet the olive oil, red wine and vegetables. In Spain the fish is an important
component of such diet. The objective of this study was to determine the dietary effects
of a protein ketogenic diet rich in olive oil, salad, fish and red wine.

Methods

A prospective study was carried out in 31 obese subjects (22 male and 19 female) with
the inclusion criteria whose body mass index and age was 36.46 ± 2.22 and 38.48 ±
2.27, respectively. This Ketogenic diet was called "Spanish Ketogenic Mediterranean
Diet" (SKMD) due to the incorporation of virgin olive oil as the principal source
of fat (≥30 ml/day), moderate red wine intake (200–400 ml/day), green vegetables and
salads as the main source of carbohydrates and fish as the main source of proteins.
It was an unlimited calorie diet. Statistical differences between the parameters studied
before and after the administration of the "Spanish Ketogenic Mediterranean diet"
(week 0 and 12) were analyzed by paired Student's t test.

Conclusion

The SKMD is safe, an effective way of losing weight, promoting non-atherogenic lipid
profiles, lowering blood pressure and improving fasting blood glucose levels. Future
research should include a larger sample size, a longer term use and a comparison with
other ketogenic diets.

Background

The international consensus is that carbohydrates are the basis of the food pyramid
for a healthy diet and that the best way to lose weight is by cutting back on calories
chiefly in the form of fat. It is generally believed that ketogenic diets may lead
to the development of several diseases. However, many studies have found that ketogenic
diets are healthier since they help to preserve muscle mass, reduce appetite, diminish
metabolic efficiency, induce metabolic activation of thermogenesis, favor increased
fat loss, promote a non-atherogenic lipid profile, lower blood pressure and decrease
resistance to insulin with an improvement in blood levels of glucose and insulin [1]. Contrary to past opinions, high carbohydrate diets may be associated with: low levels
of high-density lipoprotein cholesterol (HDLc), high levels of triacylglycerols (TG),
low-density lipoprotein cholesterol (LDLc) and total cholesterol [2], type 2 diabetes mellitus [3], metabolic syndrome, essential hypertension [4] and cancer [5].

Mediterranean diet has evident health benefits. Such diet is associated with a longer
life span [6,7] and lower rates of coronary heart disease, certain cancers [8], hypercholesterolemia, hypertension, diabetes and obesity [9]. It is difficult to define which are the healthiest constituents of the Mediterranean
diet, since it is a very varied diet that can change among the Mediterranean countries.
For example, in Spain the fish is an important component [10,11] as well as the olive oil, red wine and vegetables, that are 3 essential components
of such diet in all the countries. The healthy properties of the incorporation of
olive oil, red wine and fish consumption to a ketogenic diet could be explained by
the 3 following sections. Regarding the healthy properties of vegetables it is well
known that they are high in water, phytonutrients, antioxidants and provide a good
source of fiber.

The objective of the present study was to determine the dietary effects of the "Spanish
Ketogenic Mediterranean Diet" (SKMD). Such diet was a protein ketogenic diet under
free-living conditions with 4 important healthy components of the Mediterranean diet
in Spain: olive oil, salad, fish and red wine. Therefore, the present study was carried
out to demonstrate the changes in body weight, blood pressure, lipid profile and glucose
that might occur after the administration of SKMD throughout the period of study (12
weeks), in healthy obese subjects.

Olive oil

Olive oil, is considered the pillar of the Mediterranean diet, since it improves the
major risk factors for cardiovascular disease, such as the lipoprotein profile, blood
pressure, glucose metabolism and antithrombotic profile. Endothelial function, inflammation
and oxidative stress are also positively modulated. Some of these effects are attributed
beside the monounsaturated fatty acids (MUFA) to the minor components of virgin olive
oil [12]. Hydrocarbons, polyphenols, tocopherols, sterols, triterpenoids and other components,
despite their low concentration, non-fatty acid constituents may be of importance
because studies comparing monounsaturated dietary oils have reported different effects
on cardiovascular disease. Most of these compounds have demonstrated antioxidant,
anti-inflammatory and hypolipidemic properties [13]. Moreover, MUFA-rich diet prevents central fat redistribution and the postprandial
decrease in peripheral adiponectin gene expression and insulin resistance induced
by a carbohydrate-rich diet in insulin-resistant subjects [14].

Fish

Two long-chain Omega-3 polyunsaturated fatty acids (n-3 PUFA), eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA), are the active constituents of the fish. Low
rates of death from coronary heart disease has been report among individuals with
very high consumption of fish, although these people should limit intake of species
highest in mercury levels. Larger, longer-living predators (swordfish, shark) have
higher tissue concentrations, while smaller or shorter-lived species (anchovy, shellfish,
salmon, sardine) have very low concentrations [18].

High omega-3 consumption increases insulin sensitivity and reduces inflammatory markers
[19] and Piers et al. have hypothesized that unsaturated fats (MUFA and/or PUFA), rather
than saturated fat (SFA), are more effective in stimulating peroxisome proliferator-activated
receptor-α leading to fat oxidation, with SFA being much more readily diverted to
fat storage [20].

Methods

Subjects

A prospective study was carried out at a General Medicine Consultation (Córdoba, Spain)
in 40 overweight subjects (22 male and 19 female) whose body mass index and age was
36.46 ± 2.22 and 38.48 ± 2.27, respectively. Subjects were selected with the cooperation
of a database medical weight loss clinic. Inclusion criteria were: a diet based on
carbohydrate foods (> 50% of dairy energy intake), achievement of desired weight loss,
normal liver and renal function, not to have antecedents of gout or high uric acid,
not to have exercise, alcoholic and smoking habits, not to be pregnant or lactating,
IMC ≥ 30, age ≥ 18 years and ≤ 65 years and not to be under medication. Since obesity
increases the risk for alterations in hepatocyte function that lead to accumulation
of lipid in hepatocytes and hepatomegaly (Non-alcoholic Fatty Liver Disease), we consider
higher liver transaminase levels as a variant of normality in such obese patients
(hepatic transaminases ≤ twice normal values → GOT and GPT ≤ 80 mU/ml). Chronic hepatitis
B or C was ruled out in such patients by negative serologies. We determined normal
renal function as measured by plasma urea nitrogen and plasma creatinine: creatinine
≤ 1.3 mg/dl and urea ≤ 40 mg/dl. Subjects with the inclusion criteria were selected
for eligibility by phone and 40 eligible subjects were invited to attend an orientation
session during the week prior to the study. Patients measured their body's ketosis
state every morning by ketone strips. During the study, the participants were phoned
by the same person weekly, in order to assure the correct realization of the protocol
and the ketosis state. If the subjects failed to maintain adequate compliance with
the clinical trial protocol they would be dropped out the study.

Subjects received no monetary compensation for their participation and provided voluntary
written consent form before initiating the diet.

The Ethics and Clinical Investigation Committee of the "Spanish Medical Association
of the Proteinic Diet" approved the study protocol, informed consent form and subject
informational materials. Patient anonymity was preserved.

Diet

This protein ketogenic diet was called "Spanish Ketogenic Mediterranean Diet" (SKMD)
due to the incorporation of virgin olive oil as the principal source of fat, moderate
red wine intake, green vegetables and salads as the main source of carbohydrates and
fish as the main source of proteins. It was an unlimited calorie diet, nevertheless
subjects were encouraged to consume per day: a maximum of 30 g of carbohydrates in
the form of green vegetables and salad, a minimum of 30 ml of virgin olive oil, 200–400
ml of red wine and no limit of the protein block.

The minimum 30 ml of olive oil were distributed unless in 10 ml per principal meal
(breakfast, lunch and dinner). Red wine (200–400 ml a day) was distributed in 100–200
ml per lunch and dinner. The protein block was divided in "fish block" and "no fish
block". The "fish block" included all the types of fish except larger, longer-living
predators (swordfish and shark). The "no fish block" included meat, fowl, eggs, shellfish
and cheese. Both protein blocks were not mixed in the same day and were consumed individually
during its day on the condition that at least 4 days of the week were for the "fish
block".

Trans fats (margarines and their derivatives) and processed meats with added sugar
were not allowed.

No more than two cups of coffee or tea and at least 3 litres of water were intake
each day. Infusions and artificial sweeteners were allowed (saccharin, cyclamate,
acesulfame, aspartame and sucralose).

Measurements

Subjects were weighed and systolic/diastolic blood pressure was measurement at weeks
0, 4, 8 and 12, at the same time (that depends on the subject) and using always the
same digital scale ("Seca 703") and mercurial sphygmomanometer ("Labtron Model 03-225").

Fasting venous blood samples were collected at weeks 0 and 12 for total cholesterol,
HDLc, LDLc, triacylglycerol and glucose. Venous blood samples for glucose, lipid and
lipoprotein analysis were collected into EDTA-containing (1 g/l) tubes from all subjects
after a 12 h overnight fast at the beginning of the study and at the end of each dietary
period. Plasma was obtained by low-speed centrifugation for 15 min at 4°C within 1
h of venepuncture. Plasma cholesterol and TAG levels were determined by enzymatic
techniques. HDL-cholesterol was determined after precipitation with fosfowolframic
acid LDL-cholesterol concentration was calculated using the Friedewald formula. Plasma
glucose was measured by the glucose oxidase method. To reduce interassay variation,
plasma was stored at -80°C and analysed at the end of the study.

Statistical analysis

Statistical differences between the parameters before and after the administration
of the SKMD (week 0 and 12) were analyzed by paired Student's t test with SPSS 12.0 (SPSS Inc., Chicago, IL, USA) and are expressed as mean ± standard
error of the mean (SEM). The parameters studied were: weight, body mass index (BMI),
systolic blood pressure (SBP), diastolic blood pressure (DBP), total cholesterol,
HDLc, LDLc, triacylglycerol and glucose. Before the Student's t test, Kolmogorov-Smirnov and Shapiro-Wilk tests were used for testing normality and
the assumption of homoscedasticity was determined with the F-Snedecor test.

Results

Subject attrition

Of the 40 persons who started the study, data collected from 31 subjects were used
in the final analysis. Data were not use from 9 subjects: 3 subjects were withdrawn
for failure to maintain adequate compliance with the clinical trial protocol; 4 subjects
were lost to follow-up; 1 subject withdrew because he said the diet was too expensive;
1 subject was withdrawn due to suffer a polytraumatism car accident.

Parameters analyzed

Normal distribution and the assumption of homoscedasticity were verified. As there
were no significant differences in male and female subjects in all the parameters
examined (p > 0.05), the data of males and females in each group are pooled and presented
together. The changes in all the parameters studied are shown in Table 1.

Table 1. Changes in the level of various parameters before and after the SKMD

Discussion

Weight loss

It is thought that consumption of a high-fat-protein diet will be accompanied by a
higher weight gain. On the contrary, our results confirm that the SKMD is an effective
therapy for obesity without caloric restriction. This might be due to the fact that
there is a synergic effect between the high protein ketogenic nature of the diet and
its richness in MUFA and PUFA. We don't have data about the percentage of body fat
and lean body mass lost. Nevertheless we think that there was a more selective fat
loss because we didn't observe the flaccidity physical aspect that we have observed
before with hypocaloric diets, and subjects had a physical aspect similar to a liposuction,
since fat was removed from many different fat specific deposit areas, including the
abdomen, thighs, hips, buttocks, waist, neck and upper arms. Our hypothesis is founded
in the following statements:

1. Many studies have confirmed that the ketogenic diet is an effective therapy for
obesity [1,21-26]. In addition to the fact that an equal number of calories are ingested, ketogenic
diets are more effective for achieving fat loss than the conventional high-carbohydrate/low-fat
diets [1,26]. Low-carbohydrate diets have even proved to be more effective than conventional diets
for more selective fat loss and conserving muscle mass [1,24-26], moreover, several longer term studies have noted improvements in body composition
on a higher protein pattern despite similar weight loss [27].

3. High unsaturated fat diet is more effective to preserve lean mass than a low fat
diet or a low carbohydrate diet [23]. Moreover, the PUFA from the fish, DHA and EPA exhibit "anti-obesity" effect as well
as improving insulin sensitivity [28].

In connection with the moderate red wine consumption of the SKMD, we agree with the
statement that moderate red wine consumption (450 ml) is not associated with differences
in body weight [29], so this consumption would not affect to the weight loss.

Further trials are required to examine the potential role of the SKMD for the selective
fat loss and its protective effect against muscle protein catabolism.

Glycemic control

During the SKMD the fasting glycemia improved significantly. These findings could
be explained by the following points:

Our data are not enough to state with precision if the SKMD is the same or better
than a conventional ketogenic diet to improve glycemic control due to its higher content
in MUFA and DHA-EPA.

We think that the moderate prandial red wine consumption of the SKMD did not have
effect (beneficial or adverse) on the glycemic control, since Gin et al. reported
that moderate prandial wine consumption has no adverse effect on the glycemic control
of diabetic patients, thus it appears unnecessary to proscribe the consumption of
red wine in moderation with meals to diabetic patients [32].

Effects of the "Spanish Ketogenic Mediterranean diet" on cardiovascular parameters

The data presented in this study showed that the SKMD significantly decreases the
total cholesterol, LDLc, triacylglycerols, SBP, DBP and increases the level of HDLc.
This healthy cardiovascular profile is probably due to the favorable interaction of
the weight loss and the components of the SKMD: high protein ketogenic diet-virgin
olive oil-fish oil-red wine-salad. Our arguments are founded in the following findings:

1. Ketogenic diets improve all aspects of atherogenic dyslipidemia, decreasing fasting
and postprandial triglyceride levels and increasing HDLc and LDLc particle size [1,33]. When the ketogenic diet is higher in protein than fat, the level of LDLc also decreases
[33-35].

Explanations and Suggestions

We recognize several limitations of our study that may have influenced the study findings:

1. The sample of the study is small (31 subjects).

2. This is not a random population study, since subjects were selected for eligibility
and their eligibility was related with their compliance to the diet.

3. Weight loss may be related with improvement in all parameters that are studied.

4. We didn't take into consideration calories intake before and after the 12 weeks.
Although it is known that an equal number of calories are ingested, ketogenic diets
are more effective for achieving fat loss than the conventional high-carbohydrate/low-fat
diets [1,26], we don't know if our patients intake less food and calories, and if it is the case,
this would be correlated with weigh reduction and better cardiovascular parameters.

5. Although the effect of vitamins is not clear, especially in short interventions,
their possible contribution to better cardiovascular parameters should be possible.

6. Our study has no control groups to consider the interaction between the components
of the SKMD. There is no way to say if the healthy results are due to the ketogenic
nature of the diet, the virgin olive oil, the red wine, the higher fish intake, the
higher salad intake or a synergic effect between these components.

All these limitations should be known and accordingly considered by further trials.

Conclusion

The SKMD is safe, an effective way of losing weight, promoting non-atherogenic lipid
profiles, lowering blood pressure and improving fasting blood glucose levels. Future
research should include a larger sample size, a longer term use and a comparison with
other ketogenic diets.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

JPG was the principal researcher and was responsible for study design, acquisition
of data, analysis and interpretation of data and preparation of manuscript. AMS was
responsible for analysis and interpretation of data. AAM was responsible for study
design, analysis and interpretation of data.

Acknowledgements

The authors thank the men and women who participated in this investigation.